The long term objectives are to elucidate the molecular mechanism of signal transduction in smooth muscle, from receptor activation to contraction and relaxation, and to use this information to explore the molecular and cellular mechanism of vascular abnormality (such as hypertension) and of the regulatory processes in other cell systems. Specifically, during the proposed Project Period, we wish to further elucidate the molecular mechanism of a novel signal transduction in smooth muscle: agonist-induced, G protein-mediated inhibition of myosin light chain (MLC) phosphatase to induce the Ca2+-sensitization of MLC phosphorylation and force. We shall (1) determine, using "G protein- coupled", permeabilized smooth muscle in combination with two-dimensional gel electrophoresis, whether activation of PKC causes indirectly an activation of MLC kinase or an inhibition of the phosphatase or both to increase MLC phosphorylation and force in at a given Ca2+ concentration; (2) identify the in situ phosphorylation site(s) of MLC by PKC activation and GTPgammaS; (3) answer the question that diacylglycerol is the second messenger and resultant activation of PKC is involved in the G protein-- mediated Ca2+ sensitization. We shall further define the mechanism of G protein-mediated Ca2+-sensitization; (4) whether transient Ca2+- sensitization of MLC phosphorylation by GTPgammaS results from a re- activation of MLC phosphatase or a delayed inhibition of kinase activity; (5) to characterize the in situ MLC phosphatase regulated by G protein; (6) to examine the effects of ADP-ribosylating bacterial toxins and A1F4 to identify the type of G protein(s) responsible for the Ca2+ sensitization. We shall generalize the fact of G protein-mediated inhibition of smooth muscle MLC phosphatase; (7)to explore whether bidirectional modulation of protein phosphatase, not only inhibition but also activation, exists and has physiologically important role of cell functions and (8) to test the hypothesis that similar inhibition of protein phosphatase by G proteins has general functions in other cell systems.